RESUMO
Natural antibodies (Abs) can target host glycans on the surface of pathogens. We studied the evolution of glycan-reactive B cells of rhesus macaques and humans using glycosylated HIV-1 envelope (Env) as a model antigen. 2G12 is a broadly neutralizing Ab (bnAb) that targets a conserved glycan patch on Env of geographically diverse HIV-1 strains using a unique heavy-chain (VH) domain-swapped architecture that results in fragment antigen-binding (Fab) dimerization. Here, we describe HIV-1 Env Fab-dimerized glycan (FDG)-reactive bnAbs without VH-swapped domains from simian-human immunodeficiency virus (SHIV)-infected macaques. FDG Abs also recognized cell-surface glycans on diverse pathogens, including yeast and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike. FDG precursors were expanded by glycan-bearing immunogens in macaques and were abundant in HIV-1-naive humans. Moreover, FDG precursors were predominately mutated IgM+IgD+CD27+, thus suggesting that they originated from a pool of antigen-experienced IgM+ or marginal zone B cells.
Assuntos
Anticorpos Neutralizantes/imunologia , HIV-1/imunologia , Fragmentos Fab das Imunoglobulinas/imunologia , Polissacarídeos/imunologia , SARS-CoV-2/imunologia , Vírus da Imunodeficiência Símia/imunologia , Glicoproteína da Espícula de Coronavírus/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Animais , Linfócitos B/imunologia , Anticorpos Amplamente Neutralizantes/imunologia , COVID-19/imunologia , Dimerização , Epitopos/imunologia , Glicosilação , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/química , Macaca mulatta , Polissacarídeos/química , Receptores de Antígenos de Linfócitos B/química , Vírus da Imunodeficiência Símia/genética , Vacinas/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/química , Produtos do Gene env do Vírus da Imunodeficiência Humana/genéticaRESUMO
Peptide immunogens provide an approach to focus antibody responses to specific neutralizing sites on the HIV envelope protein (Env) trimer or on other pathogens. However, the physical characteristics of peptide immunogens can limit their pharmacokinetic and immunological properties. Here, we have designed synthetic "star" nanoparticles based on biocompatible N-[(2-hydroxypropyl)methacrylamide] (HPMA)-based polymer arms extending from a poly(amidoamine) (PAMAM) dendrimer core. In mice, these star nanoparticles trafficked to lymph nodes (LNs) by 4 hours following vaccination, where they were taken up by subcapsular macrophages and then resident dendritic cells (DCs). Immunogenicity optimization studies revealed a correlation of immunogen density with antibody titers. Furthermore, the co-delivery of Env variable loop 3 (V3) and T-helper peptides induced titers that were 2 logs higher than if the peptides were given in separate nanoparticles. Finally, we performed a nonhuman primate (NHP) study using a V3 glycopeptide minimal immunogen that was structurally optimized to be recognized by Env V3/glycan broadly neutralizing antibodies (bnAbs). When administered with a potent Toll-like receptor (TLR) 7/8 agonist adjuvant, these nanoparticles elicited high antibody binding titers to the V3 site. Similar to human V3/glycan bnAbs, certain monoclonal antibodies (mAbs) elicited by this vaccine were glycan dependent or targeted the GDIR peptide motif. To improve affinity to native Env trimer affinity, nonhuman primates (NHPs) were boosted with various SOSIP Env proteins; however, significant neutralization was not observed. Taken together, this study provides a new vaccine platform for administration of glycopeptide immunogens for focusing immune responses to specific bnAb epitopes.
Assuntos
Vacinas contra a AIDS/imunologia , HIV-1/imunologia , Nanopartículas/uso terapêutico , Animais , Anticorpos Neutralizantes/imunologia , Formação de Anticorpos/imunologia , Epitopos/imunologia , Feminino , Proteína gp120 do Envelope de HIV/química , Infecções por HIV/imunologia , Soropositividade para HIV/imunologia , Macaca mulatta , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/química , Peptídeos , PrimatasRESUMO
Viral glycoproteins are a primary target for host antibody responses. However, glycans on viral glycoproteins can hinder antibody recognition since they are self glycans derived from the host biosynthesis pathway. During natural HIV-1 infection, neutralizing antibodies are made against glycans on HIV-1 envelope glycoprotein (Env). However, such antibodies are rarely elicited with vaccination. Previously, the vaccine-induced, macaque antibody DH501 was isolated and shown to bind to high mannose glycans on HIV-1 Env. Understanding how DH501 underwent affinity maturation to recognize glycans could inform vaccine induction of HIV-1 glycan antibodies. Here, we show that DH501 Env glycan reactivity is mediated by both germline-encoded residues that contact glycans, and somatic mutations that increase antibody paratope flexibility. Only somatic mutations in the heavy chain were required for glycan reactivity. The paratope conformation was fragile as single mutations within the immunoglobulin fold or complementarity determining regions were sufficient for eliminating antibody function. Taken together, the initial germline VHDJH rearrangement generated contact residues capable of binding glycans, and somatic mutations were required to form a flexible paratope with a cavity conducive to HIV-1 envelope glycan binding. The requirement for the presence of most somatic mutations across the heavy chain variable region provides one explanation for the difficulty in inducing anti-Env glycan antibodies with HIV-1 Env vaccination.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Anti-HIV/imunologia , HIV-1/imunologia , Polissacarídeos/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS/genética , Animais , Anticorpos Neutralizantes/genética , Anticorpos Neutralizantes/imunologia , Sequência de Bases , Infecções por HIV/imunologia , Humanos , MutaçãoRESUMO
Current HIV-1 vaccines elicit strain-specific neutralizing antibodies. Broadly neutralizing antibodies (BnAbs) are not induced by current vaccines, but are found in plasma in â¼20% of HIV-1-infected individuals after several years of infection. One strategy for induction of unfavored antibody responses is to produce homogeneous immunogens that selectively express BnAb epitopes but minimally express dominant strain-specific epitopes. Here we report that synthetic, homogeneously glycosylated peptides that bind avidly to variable loop 1/2 (V1V2) BnAbs PG9 and CH01 bind minimally to strain-specific neutralizing V2 antibodies that are targeted to the same envelope polypeptide site. Both oligomannose derivatization and conformational stabilization by disulfide-linked dimer formation of synthetic V1V2 peptides were required for strong binding of V1V2 BnAbs. An HIV-1 vaccine should target BnAb unmutated common ancestor (UCA) B-cell receptors of naïve B cells, but to date no HIV-1 envelope constructs have been found that bind to the UCA of V1V2 BnAb PG9. We demonstrate herein that V1V2 glycopeptide dimers bearing Man5GlcNAc2 glycan units bind with apparent nanomolar affinities to UCAs of V1V2 BnAbs PG9 and CH01 and with micromolar affinity to the UCA of a V2 strain-specific antibody. The higher-affinity binding of these V1V2 glycopeptides to BnAbs and their UCAs renders these glycopeptide constructs particularly attractive immunogens for targeting subdominant HIV-1 envelope V1V2-neutralizing antibody-producing B cells.
Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Neutralizantes/imunologia , Glicopeptídeos/imunologia , HIV-1/imunologia , Receptores de Antígenos de Linfócitos B/metabolismo , Dimerização , Eletroforese em Gel de Poliacrilamida , Glicopeptídeos/síntese química , Glicopeptídeos/metabolismo , Ligação Proteica , Receptores de Antígenos de Linfócitos B/genética , Ressonância de Plasmônio de SuperfícieRESUMO
Critical to the search for an effective HIV-1 vaccine is the development of immunogens capable of inducing broadly neutralizing antibodies (BnAbs). A key first step in this process is to design immunogens that can be recognized by known BnAbs. The monoclonal antibody PG9 is a BnAb that neutralizes diverse strains of HIV-1 by targeting a conserved carbohydrate-protein epitope in the variable 1 and 2 (V1V2) region of the viral envelope. Important for recognition are two closely spaced N-glycans at Asn(160) and Asn(156). Glycopeptides containing this synthetically challenging bis-N-glycosylated motif were prepared by convergent assembly, and were shown to be antigenic for PG9. Synthetic glycopeptides such as these may be useful for the development of HIV-1 vaccines based on the envelope V1V2 BnAb epitope.
Assuntos
Vacinas contra a AIDS/imunologia , Antígenos/imunologia , Glicopeptídeos/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Vacinas contra a AIDS/química , Antígenos/química , Configuração de Carboidratos , Cristalografia por Raios X , Glicopeptídeos/química , Proteína gp120 do Envelope de HIV/química , Modelos Moleculares , Dados de Sequência MolecularRESUMO
Described herein is the first total chemical synthesis of the unique α-subunit of the human glycoprotein hormone (α-hGPH). Unlike the biologically derived glycoprotein hormones, which are isolated as highly complex mixtures of glycoforms, α-hGPH obtained by chemical synthesis contains discrete homogeneous glycoforms. Two such systems have been prepared. One contains the disaccharide chitobiose at the natural N-glycosylation sites. The other contains dodecamer oligosaccharides at these same sites. The dodecamer sugar is a consensus sequence incorporating the key features associated with human glycoproteins.
Assuntos
Hormônio Foliculoestimulante Humano/síntese química , Subunidade alfa de Hormônios Glicoproteicos/síntese química , Sequência de Aminoácidos , Técnicas de Química Sintética , Dissacarídeos/síntese química , Dissacarídeos/química , Hormônio Foliculoestimulante Humano/química , Subunidade alfa de Hormônios Glicoproteicos/química , Glicosilação , Humanos , Modelos Moleculares , Dados de Sequência MolecularRESUMO
This review summarizes the contribution of MALDI-TOF mass spectrometry in the study of cell-penetrating peptide (CPP) internalization in eukaryote cells. This technique was used to measure the efficiency of cell-penetrating peptide cellular uptake and cargo delivery and to analyze carrier and cargo intracellular degradation. The impact of thiol-containing membrane proteins on the internalization of CPP-cargo disulfide conjugates was also evaluated by combining MALDI-TOF MS with simple thiol-specific reactions. This highlighted the formation of cross-linked species to cell-surface proteins that either remained trapped in the cell membrane or led to intracellular delivery. MALDI-TOF MS is thus a powerful tool to dissect CPP internalization mechanisms.
Assuntos
Membrana Celular/química , Peptídeos Penetradores de Células/análise , Proteínas de Membrana/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Membrana Celular/metabolismo , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/metabolismo , Peptídeos Penetradores de Células/farmacologia , Reagentes de Ligações Cruzadas/análise , Reagentes de Ligações Cruzadas/química , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Compostos de Sulfidrila/metabolismoRESUMO
Although cell-penetrating peptides are widely used as molecular devices to cross membranes and transport molecules or nanoparticles inside cells, the underlying internalization mechanism for such behavior is still studied and discussed. One of the reasons for such a debate is the wide panel of chemically different cell-penetrating peptides or cargo that is used. Indeed the intrinsic physico-chemical properties of CPP and conjugates strongly affect the cell membrane recognition and therefore the internalization pathways. Altogether, the mechanisms described so far should be shared between two general pathways: endocytosis and direct translocation. As it is established now that one cell-penetrating peptide can internalize at the same time by these two different pathways, the balance between the two pathways relies on the binding of the cell-penetrating peptide or conjugate to specific cell membrane components (carbohydrates, lipids). Like endocytosis which includes clathrin- and caveolae-dependent processes and macropinocytosis, different translocation mechanisms could co-exist, an idea that emerges from recent studies. In this review, we will focus solely on penetratin membrane interactions and internalization mechanisms.
Assuntos
Proteínas de Transporte/química , Membrana Celular/química , Peptídeos Penetradores de Células/química , Animais , Proteínas de Transporte/metabolismo , Proteínas de Transporte/farmacologia , Membrana Celular/metabolismo , Peptídeos Penetradores de Células/metabolismo , Peptídeos Penetradores de Células/farmacologia , Endocitose/efeitos dos fármacos , Humanos , Transporte Proteico/efeitos dos fármacosRESUMO
A Diels-Alder based route to trans-fused angularly functionalized bicyclic structures has been developed. This transformation features the use of a tetrasubstituted dienophile in the cycloaddition step.
RESUMO
A highly convergent synthesis of the sialic acid-rich biantennary N-linked glycan found in human glycoprotein hormones and its use in the synthesis of a fragment derived from the beta-domain of human Follicle-Stimulating Hormone (hFSH) are described. The synthesis highlights the use of the Sinay radical glycosidation protocol for the simultaneous installation of both biantennary side-chains of the dodecasaccharide as well as the use of glycal chemistry to construct the tetrasaccharide core in an efficient manner. The synthetic glycan was used to prepare the glycosylated 20-27aa domain of the beta-subunit of hFSH under a Lansbury aspartylation protocol. The proposed strategy for incorporating the prepared N-linked dodecasaccharide-containing 20-27aa domain into beta-hFSH subunit was validated in the context of a model system, providing protected beta-hFSH subunit functionalized with chitobiose at positions 7 and 24.
Assuntos
Dissacarídeos/química , Hormônio Foliculoestimulante Humano/síntese química , Subunidade beta do Hormônio Folículoestimulante/síntese química , Ácido N-Acetilneuramínico/química , Polissacarídeos/síntese química , Dissacarídeos/síntese química , Feminino , Hormônio Foliculoestimulante Humano/química , Subunidade beta do Hormônio Folículoestimulante/química , Glicosilação , Humanos , Ácido N-Acetilneuramínico/síntese química , Polissacarídeos/químicaRESUMO
Carriers with linear or dendrimeric structures displaying different functional groups were synthesized and their delivery properties were studied.
Assuntos
Dendrímeros/química , Dendrímeros/farmacocinética , Portadores de Fármacos/química , Portadores de Fármacos/farmacocinética , Oligopeptídeos/química , Oligopeptídeos/farmacocinética , Animais , Células CHO , Cricetinae , Cricetulus , Dendrímeros/síntese química , Portadores de Fármacos/síntese química , Sistemas de Liberação de Medicamentos , Estrutura Molecular , Oligopeptídeos/síntese química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
HIV-1 envelope (Env) mimetics are candidate components of prophylactic vaccines and potential therapeutics. Here we use a synthetic V3-glycopeptide ("Man9-V3") for structural studies of an HIV Env third variable loop (V3)-glycan directed, broadly neutralizing antibody (bnAb) lineage ("DH270"), to visualize the epitope on Env and to study how affinity maturation of the lineage proceeded. Unlike many previous V3 mimetics, Man9-V3 encompasses two key features of the V3 region recognized by V3-glycan bnAbs-the conserved GDIR motif and the N332 glycan. In our structure of an antibody fragment of a lineage member, DH270.6, in complex with the V3 glycopeptide, the conformation of the antibody-bound glycopeptide conforms closely to that of the corresponding segment in an intact HIV-1 Env trimer. An additional structure identifies roles for two critical mutations in the development of breadth. The results suggest a strategy for use of a V3 glycopeptide as a vaccine immunogen.
Assuntos
Anticorpos Neutralizantes/imunologia , Produtos do Gene env/química , Produtos do Gene env/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , Motivos de Aminoácidos , Mapeamento de Epitopos , Epitopos/química , Epitopos/genética , Epitopos/imunologia , Produtos do Gene env/genética , Infecções por HIV/imunologia , HIV-1/química , HIV-1/genética , Humanos , Modelos Moleculares , MutaçãoRESUMO
We have measured the efficiencies of two novel pseudo-peptidic carriers and various cell-penetrating peptides (Penetratin, (Arg)9 and the third helix of the homeodomain of Knotted-1) to deliver the same cargo inside cells. The cargo that was studied corresponds to the pseudo-substrate of protein kinase C. Cargo delivery was quantified using a recent method based on isotope labeling and MALDI-TOF MS. Results of cargo delivery were compared to the amounts of free CPP internalized inside cells. The third helix of Knotted gave the best results concerning free CPP cellular uptake. It was also found to be the most efficient carrier. This peptide thus emerges as a new CPP with very promising properties.
Assuntos
Portadores de Fármacos/análise , Oligopeptídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Sequência de Aminoácidos , Animais , Proteínas de Transporte/análise , Proteínas de Transporte/metabolismo , Peptídeos Penetradores de Células , Células Cultivadas , Portadores de Fármacos/metabolismo , Dados de Sequência Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/metabolismoRESUMO
A preventive HIV-1 vaccine should induce HIV-1-specific broadly neutralizing antibodies (bnAbs). However, bnAbs generally require high levels of somatic hypermutation (SHM) to acquire breadth, and current vaccine strategies have not been successful in inducing bnAbs. Because bnAbs directed against a glycosylated site adjacent to the third variable loop (V3) of the HIV-1 envelope protein require limited SHM, the V3-glycan epitope is an attractive vaccine target. By studying the cooperation among multiple V3-glycan B cell lineages and their coevolution with autologous virus throughout 5 years of infection, we identify key events in the ontogeny of a V3-glycan bnAb. Two autologous neutralizing antibody lineages selected for virus escape mutations and consequently allowed initiation and affinity maturation of a V3-glycan bnAb lineage. The nucleotide substitution required to initiate the bnAb lineage occurred at a low-probability site for activation-induced cytidine deaminase activity. Cooperation of B cell lineages and an improbable mutation critical for bnAb activity defined the necessary events leading to breadth in this V3-glycan bnAb lineage. These findings may, in part, explain why initiation of V3-glycan bnAbs is rare, and suggest an immunization strategy for inducing similar V3-glycan bnAbs.
Assuntos
Anticorpos Neutralizantes/metabolismo , HIV-1/imunologia , Polissacarídeos/metabolismo , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/genética , Cristalografia por Raios X , Humanos , Masculino , Mutação/genética , Testes de Neutralização , Filogenia , Ligação Proteica , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismoRESUMO
A goal for an HIV-1 vaccine is to overcome virus variability by inducing broadly neutralizing antibodies (bnAbs). One key target of bnAbs is the glycan-polypeptide at the base of the envelope (Env) third variable loop (V3). We have designed and synthesized a homogeneous minimal immunogen with high-mannose glycans reflective of a native Env V3-glycan bnAb epitope (Man9-V3). V3-glycan bnAbs bound to Man9-V3 glycopeptide and native-like gp140 trimers with similar affinities. Fluorophore-labeled Man9-V3 glycopeptides bound to bnAb memory B cells and were able to be used to isolate a V3-glycan bnAb from an HIV-1-infected individual. In rhesus macaques, immunization with Man9-V3 induced V3-glycan-targeted antibodies. Thus, the Man9-V3 glycopeptide closely mimics an HIV-1 V3-glycan bnAb epitope and can be used to isolate V3-glycan bnAbs.
Assuntos
Anticorpos Neutralizantes/imunologia , Epitopos/imunologia , Glicopeptídeos/imunologia , HIV-1/imunologia , Mimetismo Molecular/imunologia , Animais , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/isolamento & purificação , Especificidade de Anticorpos/imunologia , Linfócitos B/citologia , Linhagem da Célula , Separação Celular , Células Clonais , Epitopos/química , Glicopeptídeos/química , Antígenos HIV/imunologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , Macaca mulatta , Domínios Proteicos , Multimerização ProteicaRESUMO
Induction of broadly neutralizing antibodies (bnAbs) that target HIV-1 envelope (Env) is a goal of HIV-1 vaccine development. A bnAb target is the Env third variable loop (V3)-glycan site. To determine whether immunization could induce antibodies to the V3-glycan bnAb binding site, we repetitively immunized macaques over a 4-year period with an Env expressing V3-high mannose glycans. Env immunizations elicited plasma antibodies that neutralized HIV-1 expressing only high-mannose glycans-a characteristic shared by early bnAb B cell lineage members. A rhesus recombinant monoclonal antibody from a vaccinated macaque bound to the V3-glycan site at the same amino acids as broadly neutralizing antibodies. A structure of the antibody bound to glycan revealed that the three variable heavy-chain complementarity-determining regions formed a cavity into which glycan could insert and neutralized multiple HIV-1 isolates with high-mannose glycans. Thus, HIV-1 Env vaccination induced mannose-dependent antibodies with characteristics of V3-glycan bnAb precursors.
Assuntos
Anticorpos Neutralizantes/imunologia , Epitopos/imunologia , Manose/imunologia , Polissacarídeos/imunologia , Primatas/imunologia , Vacinas/imunologia , Animais , Anticorpos Monoclonais/imunologia , Sítios de Ligação/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Macaca mulatta , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologiaRESUMO
We have designed a mass stable reporter (msr) tag with m/z over 500, trifluoroacetyl(alpha,alpha-diethyl)Gly-Lys(Nepsilonbiotin)-(D)Lys-Cys, for the quantification of the uptake and study of the degradation processes of cell-penetrating peptides (CPP), by matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. This tag was found stable in cell lysis conditions. Using a quantitative MALDI-TOF mass spectrometry analysis based method, an accurate tracking of a new CPP and of its degradation products could be done. (1) The new msr(W/R) nonapeptide (H-RRWWRRWRR-NH2) enters chinese hamster ovary (CHO) K1 cells with a kinetic reaching a steady state after 30-60 min of incubation. This plateau was stable for 4 h and decreased slowly afterward. (2) The peptide msr(W/R) nonapeptide was not cytotoxic over 48 h incubation with CHO cells. (3) After 1 h incubation, the msr(W/R) nonapeptide accumulated with a 3-fold higher concentration than the extracellularly added concentration (7.5 microM). (4) The intracellular quantification was accurate with less than 3% of the quantified peptide being potentially membrane-bound. (5) There was no leakage of the full-length CPP outside the cells. And, finally, (6) analysis of the degradation process of this new CPP suggests that the peptide did not traffick to lysosomes.
Assuntos
Biotina/análogos & derivados , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Biotina/química , Biotina/metabolismo , Células CHO , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Cricetinae , Cricetulus , Lisossomos/enzimologia , Oligopeptídeos/análise , Potássio/farmacologia , Transporte ProteicoRESUMO
Trojan peptides or cell-penetrating peptides (CPP) are natural or designed peptides identified as cellular membrane-crossing molecules, in particular through their potency to vehiculate various kinds of compounds to the cytoplasm and nucleus of living cells. The indirect methods used so far to detect these peptides in cells led to controversial hypotheses on the mechanism of their cell entry. Therefore, we have developed a MALDI-TOF mass spectrometry-based quantification method to track these peptides inside cells. This new method is presented in this review.